Studies regarding production of industrial products such as biodiesel fuel and/or food products by using, as raw materials, compounds including fatty acid produced by unicellular photosynthetic organisms (hereinafter referred to as “microalgae”) or TAG that releases fatty acid as a result of hydrolysis (which are collectively referred to as “lipids”), have been conducted over the world. However, under the current circumstances, lipid production costs are high, and thus, it is difficult to produce biodiesel fuel and the like on the commercial basis (Non Patent Literature 1). Hence, further technical developments for reducing the production costs of biodiesel fuel and the like have been continued, and one of such technical developments is a study regarding the improvement of the TAG productivity of microalgae.
A majority of microalgae accumulate a part of assimilation products generated as a result of photosynthesis in the form of starch or TAG. The accumulated amounts of such starch and TAG are different depending on the types of microalgae, and are also different depending on culture conditions although they are produced from the same type of organism. This is considered because the speed of converting photosynthetic assimilation products to starch and TAG and the speed of decomposing once synthesized storage substances are different, depending on organism species and culture conditions, and because it appears as a difference in the accumulated amounts of starch and TAG.
Raw materials for TAG are glycerol-3-phosphate and fatty acid. Glycerol-3-phosphate is synthesized from glycerol and ATP as substrates, by the action of glycerol kinase. On the other hand, fatty acid is biosynthesized in a chloroplast.
The initial reaction of fatty acid biosynthesis is catalyzed by acyl-CoA carboxylase, and malonyl-CoA is produced from acetyl-CoA. Malonyl-CoA reacts with an acyl carrier protein (ACP) to produce malonyl-ACP. Malonyl-ACP reacts with acyl-ACP prepared by binding an acyl group with ACP (C=2: acetyl-ACP, C=4: butyryl-ACP, etc.) to extend two carbon chains of acyl-ACP. When this extension reaction is repeated so that the length of carbon chains of acyl groups becomes (most commonly) 16, the acyl-ACP is hydrolyzed to palmitic acid (C16:0) and ACP by the action of acyl-ACP thioesterase. Palmitic acid binds to CoA to become palmityl-CoA, and the palmityl-CoA transfers from the chloroplast to endoplasmic reticulum. Very long chain fatty acid elongase and fatty acid desaturase further act on the palmityl-CoA, so as to produce a CoA ester of oleic acid that is monovalent unsaturated fatty acid (oleyl-CoA:C18:1). Palmitic acid and oleic acid are fatty acids that are contained in the highest contents in many organisms.
Biosynthesis of TAG is carried out on the endoplasmic reticulum membrane. First, by the action of glycerol-3-phosphate acyltransferase, an acyl group of acyl-CoA is added to the sn-1 position of glycerol-3-phosphate, so as to generate lysophosphatidic acid. Subsequently, by the action of acylglycerophosphate acyltransferase, an acyl group of acyl-CoA is added to the sn-2 position of lysophosphatidic acid, so as to generate phosphatidic acid. Phosphatidic acid is dephospharylated by phosphatidate phosphatase, and is converted to diacylglycerol. Thereafter, by the action of diacylglycerol acyltransferase (DGAT), TAG is synthesized from the diacylglycerol. DGAT is classified into two families each having a different evolutionary origin, and thus, the two DGATs are referred to as DGAT1 and DGAT2, respectively. This pathway of synthesizing TAG from glycerol-3-phosphate and acyl-CoA is referred to as a Kennedy pathway for TGA synthesis.
TAG is also synthesized by reactions other than this Kennedy pathway. An example is the following reaction involving phospholipid:diacylglycerol acyltransferase:Phospholipid+1,2-diacylglycerol=lysophospholipid+TAG
Several studies have already been conducted to increase enzyme activity associated with the TAG synthesis according to a genetic recombination technique, and to improve the amount of TAG produced per unit time and per unit light-receiving area of microalgae (hereinafter referred to as “TAG productivity”), so as to contribute to a reduction in the biodiesel production costs.
A large number of reports have been made regarding that TAG productivity is increased by allowing a gene encoding DGAT that is the last enzyme in the above-described Kennedy pathway to express at a high level. For example, by introducing a DGAT gene derived from the Gram-negative bacterium Acinetobacler into a Synechococcus elongatus PCC 7942 strain belonging to Cyanobacteria, the Synechococcus became to accumulate approximately two times the amount of TAG (Patent Literature 1).
Patent Literature 2 discloses that a gene encoding DGAT, phospholipid: diacylglycerol acyltransferase or phosphatidic acid phosphatase is introduced into Nannochloropsis belonging to Heterokontophyta, Eustigmataceae, so as to increase the content of TAG. However, this patent literature does not describe details of the effects of such gene introduction in the Examples thereof.
A DGAT1 gene derived from various organisms having a Pleckstrin-homology domain was introduced into Nannochloropsis, and as a result, the content of lipids in the transformed strain was increased in comparison to that in a wild-type strain (Patent Literature 3).
A mouse-derived DGAT was introduced into Nannochloropsis, and as a result, the content of TAG was increased (Patent Literature 4).
The above-mentioned plurality of DGAT2 genes (homologous genes) are present in Chlamydomonas reinhardtii belonging to Viridiplantae, Chlorophyta (hereinafter referred to as “green algae”). Although these homologous genes were allowed to highly express in Chlamydomonas reinhardtii, an increase in the content of lipids was not observed. On the other hand, when one of the DGAT2 homologous genes of Chlamydomonas reinhardtii was allowed to express in yeast, the transformed yeast exhibited higher TAG productivity than the wild-type yeast (Non Patent Literature 2).
When DGAT2 of Phaeodactylum tricornutum belonging to Heterokontophyta, Bacillariophyceae was cloned and the obtained clones were then allowed to highly express in the same strain, the TAG productivity of this strain was increased (Non Patent Literatures 3 and 4).
A gene encoding the initial enzyme of the Kennedy pathway for TAG synthesis, glycerol-3-phosphate acyltransferase, was separated from the green algae Lobosphaera incisa, and thereafter, it was introduced into Chlamydomonas reinhardtii and was allowed to express therein, and as a result, TAG productivity was significantly increased (Non Patent Literature 5).
Moreover, glycerol kinase that synthesizes glycerol-3-phosphate as a substrate of the above-described glycerol-3-phosphate acyltransferase was allowed to highly expressed in the diatom Fistulifera solaris JPCC DA0580 strain, and as a result, TAG productivity was slightly increased (Non Patent Literature 6).
On the other hand, it has been reported that, when an acetyl-CoA carboxylase gene and an acyl-ACP thioesterase gene that are initial enzymes for fatty acid synthesis are allowed to highly expressed in Escherichia coli, the content of free fatty acid is increased (Non Patent Literature 7).
However, in many studies, such an acyl-ACP thioesterase gene has been used to promote the synthesis of, not fatty acids (C16 and C18) having a carbon chain with a common length, but relatively short fatty acids (C10, C12 and C14). It has been clarified that lipids including fatty acids such as C10 or C12 are accumulated in seeds of the plant of the family Lauraceae, Umbellularia californica, and that such accumulation is caused by acyl-ACP thioesterase that hydrolyzes acyl-ACP having a C12 carbon chain of this plant. When the cDNA of the acyl-ACP thioesterase gene of U. californica having the substrate specificity of hydrolyzing relatively short fatty acids was introduced into Escherichia coli and was allowed to highly express therein, free fatty acids of C12 and C14 were synthesized and were then discharged to outside of the cells (Non Patent Literature 8).
Studies regarding that a gene encoding acyl-ACP thioesterase and a gene encoding diacylglycerol acyltransferase (DGAT2) are introduced into the green algae Chlamydomonas reinhardtii, so as to increase the rate of the C12 fatty acid have been disclosed (Patent Literature 5). However, the accumulation of TAG per dry weight of this recombinant was found to be at maximum approximately 2%, and even if free fatty acids were included, the accumulation of TAG was 7% or less.
By the way, Pseudococcomyxa sp. KJ strain belonging to the green algae Trebouxiophyceae (hereinafter referred to as “KJ strain”) is unicellular green algae having extremely high TAG productivity, which has been separated from hot spring water (Patent Literature 6), and the KJ strain can be cultured in the open culture system disclosed in Patent Literature 7.